Genetic evidence has shown that a germ-line deletion of chromosome 13 in the region q14.1 is associated with the development of retinoblastoma. The fact that the aberrant chromosome 13 is often reduced to a state of homozygosity or hemizygosity in some retinoblastoma suggest that a recessive mutation of a gene (or genes) within 13q14.1 contributes directly to tumorigenesis. The main objective of this proposal is to isolate this putative suppressor gene (Rb) and to study its function. The approach of chromosomal walking with DNA probes in the vicinity of the Rb gene will be used for its isolation. To obtain DNA probes in the vicinity of the Rb gene, large numbers of the random unique clones will be isolated from a chromosome 13 library. These clones will then be selected for their close proximity to the Rb gene by hybridization to Southern blots of a panel of retinoblastoma DNA with known deletions in the 13q14.1 region. Probes in the deletion region will be used as starting points for chromosomal walking. A cosmid library with specific features to facilitate chromosomal walking will be constructed. The end point of walking will be when one or more submicroscopic deletion regions is covered by overlapping clones. Moreover, the majority of retinoblastomas will show structural abnormality (deletion, translocation or altered restriction fragment lengths in this region). DNA probe from these overlapping clones will be used to screen a cDNA library for gene(s) in this region. The Rb gene(s) will be identified as the one whose mRNA expression is absent from the majority of our collection of retinoblastomas. Once the Rb gene(s) is cloned, it will be characterized structurally by: (1) DNA sequencing of the cDNA clone and the 5' flanking genomic sequences. (2) Restriction mapping of the genomic clones. The Rb gene probe will be used: (1) To examine structural features of the chromosomal aberrations in retinoblastoma. (2) To examine whether the same gene (rb) is involved in tumors such as osteosarcoma and fibrosarcoma which are clinically related to retinoblastoma. Antibodies against the Rb gene products will be raised to examine expression of the Rb gene at the protein level in normal as well as tumor cells.